Cellulase synthesis in 2-deoxy glucose resistant mutants of trichoderma reesei p-2
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Date
2005
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Publisher
CCSHAU
Abstract
A study was under taken to isolate 2-DG resistant mutants of
Trichoderma reesei P-2 for cellulase synthesis. Spores of T. reesei
were mutagenized with UV irradiation for a time period of 15
minutes. At this time maximum number of mutants were
obtained.Antimetabolite resistant mutants were obtained on 0.05%
2-DG containing Reese medium plates. A total of 24 isolates which
grew profusely, were purified and again patched on Reese medium
contained 2-DG. Finally 11 mutants showed good growth on this
medium. 2-DG resistant mutants of T. reesei mutants MU-22 and
MU-24 showed maximum cellulase production and selected. T.reesei
mutants MU-22 and MU-24 showed maximum FPase activity (1.54
IU/ml and 1.56 IU/ml) respectively, CMCase activity (6.4IU/ml and
5.5IU/ml) respectively in cellulose medium. However and
-glucosidase activity was more in MU-22(0.28 IU/ml). Effect of
different carbon sources on synthesis of cellulases were analysed in
mutants MU-24 and MU-22. Cellulose was found to be the best
carbon source whereas in cellobiose and cotton medium, cellulase
activity was reduced. In phosphocellulose medium -glucosidase
activity was high and sorbitol was not a good inducer for the
synthesis of cellulases. The optimization studies for cellulase
production by MU-24 showed that cellulose at 1% concentration
produced high level of cellulases. Similarly ammonium sulphate at a
concentration of 0.14% was found to be essential for all the three
enzyme activity. Since T. reesei is a mesophilic organism, at high
temperature it did not grow. The optimum temperature for growth
of T. reesei was found to be 30ºC. Saccharification of rice straw
indicated the release of 50% reducing sugar which were more
(35%) then the parents strain. The protein profile of culture filtrate
of T. reesei mutants were determined by SDS PAGE. It was
observed that the first set of proteins which appeared during
cellulase induction were of 50KDa, 40KDa, 30KDa and 25KDa.
Thus from this investigation it can be concluded that level of
exoglucanase, endoglucanase and -glucosidase has been altered by
mutation and such improvement in altering the level of individual
component of cellulase complex is practically possible.